**4. Regulation of the use of LAB**

Streptomycin was the first aminoglycoside reported for which resistance appeared in enterococcal strains (concentrations higher than 2000 μg/mL); this resistance is carried out by adenylation of streptomycin, by the action of the enzyme streptomycin adenyltransferase, encoded by the *aadA* gene [35, 41]. Resistance to gentamicin, kanamycin, neomycin, and netilmicin (aminoglycosides as well) is mainly due to the production of the bifunctional enzyme 2′-phosphotransferase-6′ acetyltransferase, which promotes the ATP-dependent phosphorylation of aminoglycosides [41]. Strains of enterococci of clinical origin between 60 and 65% exhibit resistance to tetracyclines, although these antibiotics are not routinely used in the treatment of infections caused by these microorganisms. There are two fundamental mechanisms of resistance to tetracyclines in enterococci: flow pumps and protection of the ribosome, thus preventing the binding of the antibiotic. The *tetK* and *tetL* genes code for proteins associated to flow pumps responsible to remove the antibiotic outside of the cell, while the *tetM*, *tetO*, and *tetS* genes code for proteins that provide resistance to tetracyclines for ribosome protection. The *tetL* and *tetM* genes are the most frequent in the chromosome and mobile determinants [41, 52, 53]. Finally, vancomycin (glycopeptide) is the main cause of concern, since this antibiotic is considered at the last option for antibiotic therapy for the treatment of Gram-positive bacteria. The resistance to vancomycin in enterococci is varied, having described six genotypes called *vanA*, *vanB*, *vanC*, *vanD*, *vanE,* and *vanG,* where the genotype *vanA* is more frequent in the *Enterococcus*

In general, Lactobacilli have a high natural resistance to vancomycin, bacitracin, cefoxitin, metronidazole, nitrofurantoin, and sulfadiazine, as well as antibiotics that inhibit the synthesis of proteins such as chloramphenicol, erythromycin, quinupristin/dalfopristin, lincomycin, clindamycin, and tetracyclines [45]. Guo et al. [54] observed 85% of incidence of vancomycin resistance in food isolated *Lactobacillus* strains, especially in *Lactobacillus plantarum* and *Lactobacillus casei*, with the lower frequency for *Lactobacillus helveticus*, but these resistances are not transferable, as genes are located in the chromosome [54]. In addition, genes that code for resistance to tetracycline and erythromycin have been detected in different *Lactobacillus*

The genus *Lactobacillus* is an excellent receptor for exogenous genes by conjugation, as demonstrated by Abriouel et al. [45] for the conjugative pAMβ1 plasmid found in *Lactobacillus plantarum* that could be obtained from enterococci and streptococci. *Lactobacillus* are commonly susceptible to antibiotics, such as penicillins (ampicillin, oxacillin, and piperacillin), inhibitors of β-lactamase, and cephalosporins (cephalothin and cefuroxime, ceftriaxone and cefoxitin), but in recent years some authors have reported resistance to penicillin G in some strains of *Lactobacillus rhamnosus*, *Lactobacillus reuteri*, and *Lactobacillus plantarum* [45, 56]. Other studies demonstrated that *Lactobacillus rhamnosus* is safe to use as a starter or probiotic culture, despite having resistance genes to vancomycin, as these resistance is encoded into the chromosome [45, 48, 54].

The horizontal gene transfer (HGT) involves the gene interchange between different bacteria through mobile DNA elements such as plasmids, conjugative transposons, integrons, and

genus [41].

*3.2.2. Lactobacillus*

64 Antimicrobial Resistance - A Global Threat

species isolated of probiotics and foods [12, 31, 55].

**3.3. Horizontal transfer of LAB to the intestinal microbiota**

The FDA categorizes microorganisms with the GRAS distinction after being evaluated in general aspects of safety, taxonomy, potential to produce pathogenicity toxins, resistance to antibiotics, and the historical background of food safety. LAB have a broad history of use in fermented foods and usually recognized as safe. However, the dissemination of AR genes puts the GRAS category in another context, especially for bacteria that present mobile genes of transfer such as *Lactobacillus*, since in the US there are still no guidelines that contemplate the type of resistance in microorganism used in food processing [57]. On the other hand, the EU commission regulates the safety of LAB used as starter or probiotic cultures in the European continent, through the EFSA that establishes guidelines for assigning qualified presumption of safety quality to the organisms since 2003. As previously mentioned, the term QPS is based on reasonable and qualified evidence to allow certain restrictions and may be analogous to the GRAS concept but with more rigid guidelines in which the reliable safety of the bacteria is verified, making clear the phrase "from farm to fork" [58]. The QPS status is given to a bacterium, by the EFSA BIOHAZ Panel (Biological Hazards) that must take into account the following aspects (**Figure 3**): (1) the identity of the taxonomic unit at the genus level; (2) documentation related to the LAB safety, based on scientific evidence and history of use; (3) pathogenicity, in which it is evaluated if any species of the genus has pathogenicity factors, if the information is available, the pathogenic strains are excluded; and (4) knowledge of the final use of the microorganism, identifying if the bacteria is part of the food chain or if it is used to produce other products [6, 58].

The list of QPS includes species of *Lactobacillus sakei*, *Lactobacillus curvatus*, *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Lactobacillus brevis*, *Lactobacillus rhamnosus*, *Lactobacillus* 

a complementary technique involves the search for AR genes using PCR techniques and microarrays [25, 29, 54]. Also, identifying the location of these genes allows to determine their potential transfer, while their sequencing can provide evidence of their bacterial taxa and

Antibiotic Resistance in Lactic Acid Bacteria http://dx.doi.org/10.5772/intechopen.80624 67

Functional metagenomics is an important approach in the investigation of antibiotic resistance genes (ARG) since it can be used to identify and characterize new ARG, including those not previously associated with antibiotic resistance [48, 61]. It is also one of the most recent techniques in the study of resistance in pure bacterial groups or more complex samples such as food; some works reported in the literature indicate the wide diversity of resistance systems that are present in food, considering the cultivable and not cultivable bacteria. Metagenomic studies help to understand the mechanisms of resistance in such a way that it allows direct applications in the identification of new drugs and the synthesis of novel and active antibiotic molecules [61].

The FEEDAP Panel proposed a scheme to evaluate the resistance present in lactic acid bacteria that can be used as probiotic or starter cultures in food processing; as previously mentioned, it is essential to distinguish between the intrinsic and acquired resistance as part of the food safety of lactic acid bacteria [58, 62]. The correct identification of the bacteria (sequencing and comparison of the 16S rDNA gene in international databases) by molecular taxonomy is essential to evaluate the type of resistance, since the intrinsic resistance is specific for a specie or genus. Once the specie under study has been identified, the MIC (minimum inhibitory concentration)

**Figure 4.** Proposed scheme for the antibiotic resistance assessment of lactic acid bacteria used as probiotic and starter

culture. Adapted from Laulund et al. [58] and EFSA [62].

identity of the genes, which helps to trace the origin of their genomes [29].

**5.1. Procedure to evaluate LAB resistant to antibiotic used in food**

**Figure 3.** Scheme for assessing the suitability for qualified presumption of safety (QPS) status of a BAL. adapted from Laulund et al. [58].

*alimentarius*, *Leuconostoc lactis, Leuconostoc citreum*, *Leuconostoc mesenteroides*, *Leuconostoc pseudomesenteroides*, *Pediococcus acidilactici*, *Pediococcus dextrinicus*, *Pediococcus pentosaceus*, *Lactococcus lactis*, and *Streptococcus thermophilus* [6]. In the case of *Enterococcus*, the QPS category cannot be assigned to all species; each specie must be individually analyzed [6].

### **5. Methods to identify antibiotic-resistant LAB**

Most widely used antibiotic susceptibility testing methods are based on (1) phenotypic detection of antibiotic resistance by measuring bacterial growth in the presence of the tested antibiotic and (2) molecular identification of resistant genotypes through polymerase chain reaction (PCR) [21, 25, 29, 39, 54]. The evaluation of phenotypic susceptibility to antibiotics in lactic acid bacteria should be done using recognized methods that allow the identification of the minimum inhibitory concentration (MIC) for the most commonly used antibiotics. Most LAB species used in food can be evaluated by the method described in ISO 10932: 2010 [59], considering the conditions and culture media for *Bifidobacteria* and LAB that do not belong to the genus enterococci [56, 57]. In case of having strains of *Enterococcus*, it is recommended to use the methods described by the Clinical and Laboratory Standards Institute [21, 60]. Some of the recommended methods to determine the MIC in LAB are the E-test, the Kirby-Bauer test (diffusion method), and the broth microdilution method (MDIL) [43]. In particular the cutoff values are known for the genera *Lactobacillus, Pediococcus*, *Lactococcus*, *Streptococcus*, and *Bifidobacteria*. The MDIL method is widely used to evaluate MIC for a large number of strains and antibiotics, although the method has some limitations, especially for those antibiotics for which a strain could quickly acquire resistance [43]. However, MIC evaluation in LAB is somewhat inconsistent among the researchers, mainly due to the lack of culture media that can ensure proper growth of LAB without interfering with the assay results. Therefore, a complementary technique involves the search for AR genes using PCR techniques and microarrays [25, 29, 54]. Also, identifying the location of these genes allows to determine their potential transfer, while their sequencing can provide evidence of their bacterial taxa and identity of the genes, which helps to trace the origin of their genomes [29].

Functional metagenomics is an important approach in the investigation of antibiotic resistance genes (ARG) since it can be used to identify and characterize new ARG, including those not previously associated with antibiotic resistance [48, 61]. It is also one of the most recent techniques in the study of resistance in pure bacterial groups or more complex samples such as food; some works reported in the literature indicate the wide diversity of resistance systems that are present in food, considering the cultivable and not cultivable bacteria. Metagenomic studies help to understand the mechanisms of resistance in such a way that it allows direct applications in the identification of new drugs and the synthesis of novel and active antibiotic molecules [61].

#### **5.1. Procedure to evaluate LAB resistant to antibiotic used in food**

*alimentarius*, *Leuconostoc lactis, Leuconostoc citreum*, *Leuconostoc mesenteroides*, *Leuconostoc pseudomesenteroides*, *Pediococcus acidilactici*, *Pediococcus dextrinicus*, *Pediococcus pentosaceus*, *Lactococcus lactis*, and *Streptococcus thermophilus* [6]. In the case of *Enterococcus*, the QPS cat-

**Figure 3.** Scheme for assessing the suitability for qualified presumption of safety (QPS) status of a BAL. adapted from

Most widely used antibiotic susceptibility testing methods are based on (1) phenotypic detection of antibiotic resistance by measuring bacterial growth in the presence of the tested antibiotic and (2) molecular identification of resistant genotypes through polymerase chain reaction (PCR) [21, 25, 29, 39, 54]. The evaluation of phenotypic susceptibility to antibiotics in lactic acid bacteria should be done using recognized methods that allow the identification of the minimum inhibitory concentration (MIC) for the most commonly used antibiotics. Most LAB species used in food can be evaluated by the method described in ISO 10932: 2010 [59], considering the conditions and culture media for *Bifidobacteria* and LAB that do not belong to the genus enterococci [56, 57]. In case of having strains of *Enterococcus*, it is recommended to use the methods described by the Clinical and Laboratory Standards Institute [21, 60]. Some of the recommended methods to determine the MIC in LAB are the E-test, the Kirby-Bauer test (diffusion method), and the broth microdilution method (MDIL) [43]. In particular the cutoff values are known for the genera *Lactobacillus, Pediococcus*, *Lactococcus*, *Streptococcus*, and *Bifidobacteria*. The MDIL method is widely used to evaluate MIC for a large number of strains and antibiotics, although the method has some limitations, especially for those antibiotics for which a strain could quickly acquire resistance [43]. However, MIC evaluation in LAB is somewhat inconsistent among the researchers, mainly due to the lack of culture media that can ensure proper growth of LAB without interfering with the assay results. Therefore,

egory cannot be assigned to all species; each specie must be individually analyzed [6].

**5. Methods to identify antibiotic-resistant LAB**

Laulund et al. [58].

66 Antimicrobial Resistance - A Global Threat

The FEEDAP Panel proposed a scheme to evaluate the resistance present in lactic acid bacteria that can be used as probiotic or starter cultures in food processing; as previously mentioned, it is essential to distinguish between the intrinsic and acquired resistance as part of the food safety of lactic acid bacteria [58, 62]. The correct identification of the bacteria (sequencing and comparison of the 16S rDNA gene in international databases) by molecular taxonomy is essential to evaluate the type of resistance, since the intrinsic resistance is specific for a specie or genus. Once the specie under study has been identified, the MIC (minimum inhibitory concentration)

**Figure 4.** Proposed scheme for the antibiotic resistance assessment of lactic acid bacteria used as probiotic and starter culture. Adapted from Laulund et al. [58] and EFSA [62].

in which the LAB is sensitive to the antibiotic analyzed is determined. The bacterium can be considered safe when the MIC is lower than the cutoff level (MIC < cutoff). On the other hand, if the MIC value is above the cutoff value (MIC > cutoff), the bacterium is considered resistant to the antibiotic, and its resistance should be confirmed by molecular methods as PCR [39, 54, 62]. However, the resistance genes not always are expressed but can be transferred to other bacteria if the environmental conditions stimulate the expression of these genes [34]. If the bacteria have intrinsic resistance, it is considered acceptable for use in food. Otherwise, it must be demonstrated whether the acquired resistance is in mobile genetic material or was acquired in the process of mutation in the bacterial chromosome (also acceptable for use in foods). Finally, the bacteria are not accepted by any regulatory body for its application in food if it is demonstrated that the resistance is exogenous and easily transferable (**Figure 4**).

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